1. Field of the Invention
The present invention relates to a hybrid vector system which incorporates elements of herpesvirus and adeno-associated virus and which is capable of expressing a gene product in eukaryotic cells. The vector system of the present invention provides a means of packaging plasmid DNA into highly infectious virions which can efficiently and safely deliver large transgenes to both mitotic and postmitotic cells in a state which can be maintained for extended periods. The invention also pertains to the use of this vector system in introducing and expressing gene sequences in mitotic and postmitotic cells for therapeutic purposes.
2. Related Art
A variety of vectors have been developed for gene delivery to the nervous system and to brain tumors. These vectors derive from herpes simplex virus type 1 (HSV-1), adenovirus, adeno-associated virus (AAV) and retrovirus constructs (for review see Friedmann, T., Trends Genet 10:210-214 (1994); Jolly, D., Cancer Gene Therapy 1 (1994); Mulligan, R. C., Science 260:926-932 (1993); Smith, F. et al., Rest. Neurol. Neurosci. 8:21-34 (1995)). Vectors based on HSV-1, including both recombinant virus vectors and amplicon vectors, as well as adenovirus vectors can assume an extrachromosomal state in the cell nucleus and mediate limited, long term gene expression in postmitotic cells, but not in mitotic cells. HSV-1 amplicon vectors can be grown to relatively high titers (10.sup.7 transducing units/ml) and have the capacity to accommodate large fragments of foreign DNA (at least 15 kb, with 10 concatemeric copies per virion). AAV vectors (rAAV), available in comparable titers to amplicon vectors, can deliver genes (&lt;4.5 kb) to postmitotic, as well as mitotic cells in combination with adenovirus or herpes virus as helper virus. Long term transgene expression is achieved by replication and formation of "episomal" elements and/or through integration into the host cell genome at random or specific sites (for review see Samulski, R. J., Current Opinion in Genetics and Development 3:74-80 (1993); Muzyczka, N., Curr. Top. Microbiol. Immunol. 158:97-129 (1992)). HSV, adenovirus and rAAV vectors are all packaged in stable particles. Retrovirus vectors can accommodate 7-8 kb of foreign DNA and integrate into the host cell genome, but only in mitotic cells, and particles are relatively unstable with low titers. Recent studies have demonstrated that elements from different viruses can be combined to increase the delivery capacity of vectors. For example, incorporation of elements of the HIV virion, including the matrix protein and integrase, into retrovirus vectors allows transgene cassettes to enter the nucleus of non-mitotic, as well as mitotic cells and potentially to integrate into the genome of these cells (Naldini, L. et al., Science 272:263-267 (1996)); and inclusion of the vesicular somatitis virus envelope glycoprotein (VSV-G) increases stability of retrovirus particles (Emi, N. et al., J. Virol. 65:1202-1207 (1991)). As another example, inclusion of elements from Epstein Barr virus (EBV)--the DNA origin of replication, oriP, and the EBNA-1, within HSV amplicon vectors allows nuclear replication of vectors in dividing human cells (Wang and Vos, in press). Still there is a need for a safe, high titer vector with a broad host range which has a large transgene capacity and can confer stable expression in both mitotic and non-mitotic cells.